The present invention generally relates to nano-electrospray emitters, nano-electrospray devices containing such emitters, methods of making such emitters, and use thereof.
Electrospray ionization (xe2x80x9cESIxe2x80x9d) (Whitehouse et al., 1985; Meng et al., 1988) has revolutionized the use of mass spectrometry in bioanalytical chemistry because of its ability to transfer large macromolecules from solution into the gas-phase as intact multiply-charged molecular ions. A special advantage of ESI is the ease with which it may be coupled to liquid chromatography (xe2x80x9cLCxe2x80x9d) (Banks, 1995), capillary electrophoresis (xe2x80x9cCExe2x80x9d) (Smith et al., 1993), and capillary electrochromatography (xe2x80x9cCECxe2x80x9d) (Schmeer et al., 1994). In the last five years, a number of research groups have developed methods for decreased sample consumption in ESI by using much lower flow rates (nL/min) than with conventional ESI (xcexcL/min) (Emmett and Caprioli, 1994; Kriger et al., 1995; Valaskovic et al., 1995a; Kelly et al., 1997; Wilm and Mann, 1996). Of these low flow ESI methods, the flow rate is controlled by some type of pump in microspray (Emmett and Caprioli, 1994), whereas in nanospray the flow rate is controlled by the potential difference between the emitter and counter-electrode (Wilm and Mann, 1996). Typically, nanospray has been accomplished by pulling silica or glass substrates under heat to produce tapered emitters with small inner diameters, e.g., a few xcexcm. Attomole level sample consumption has been achieved using nanospray (Valaskovic et al., 1995a; Valaskovic et al., 1996a).
While nanospray provides an avenue to achieve low-level detection limits with MS using only a few xcexcL of sample, even at high salt and/or buffer concentrations, most nanoliter-flow ESI emitters suffer from short operating lifetimes, poor durability, and/or low reproducibility. For example, metallized coatings have been applied to emitter substrates to provide electrical contact at the ESI outlet, but such emitters are highly susceptible to deterioration by electrical discharge (Valaskovic et al., 1995a).
To overcome this serious limitation, methods to increase metallized emitter lifetime in nanospray have been developed. These include chemical derivatization of an organofunctional silane on the emitter substrate prior to metallization (Kriger et al., 1995), by depositing a SiOx overcoating atop the metallized layer (Valaskovic et al., 1996b), or by controlled electrochemical deposition of metal film onto the emitter substrate (Kelly et al., 1997). Though these multi-layer and electrolysis methods have shown improvement in emitter durability for low flow ESI, they are tedious and time-consuming. Alternatively, nonmetallized emitters have been employed with low flow ESI by remotely coupling the ESI voltage to the emitters (Emmett et al., 1998; Hannis and Muddiman, 1998). A disadvantage of this approach, however, is that it relies on the conductive properties of the solution used, rather than the conductive properties of the emitter itself. Solution conditions may vary widely for both LC and capillary separations, and such emitters may exhibit wide differences in performance depending upon the mobile phase conditions used in the separation. Placing a metal wire into the tip for electrical contact has proved beneficial for durability (Kelleher et al., 1997; Cao and Moini, 1997; Fong and Chan, 1999), however it would be preferable to avoid the need to regulate the insertion, removal, or adjustment of an independent metal wire.
As stated by Lausecker et al., a good, long-acting and stable electrical contact on the CZE capillary terminus remains the main challenge (Lausecker et al., 1998) in performing MS coupled to capillary separations. Thus, to fully exploit the advantages of nanospray-MS with CE for the purposes of molecular species identification of analytes in biological fluids (i.e., at sub-picomole levels), a stable and/or reproducible type of nanospray emitter must be developed.
For nanospray ESI-MS emitters to be useful in coupling to either CE or CEC, the emitters must remain stable throughout the separation process. Failure of the emitter during the course of the separation is not acceptable. For quantification in particular, if calibration curves of multiple analytes at multiple concentration levels are to be constructed, single emitters with longer lifetimes or multiple emitters showing reproducible performance and ionization efficiency are needed.
The present invention is directed to overcoming these deficiencies in the art.
One aspect of the present invention relates to a nanospray emitter including an emitter body which includes a fluid inlet, an outlet orifice, and a passage communicating between the fluid inlet and outlet orifice; and an electrically conductive polymer coating on at least a portion of the emitter body.
A further aspect of the present invention relates to a nano-electrospray device including a counter-electrode and a nanospray emitter of the present invention whose inlet is capable of fluid communication with a fluid source including a fluid containing one or more analytes. Either the counter-electrode or the nanospray emitter can be either coupled to a power supply, with the other being grounded, for development of a suitable electrical potential between the counter-electrode and the nanospray emitter, which causes the fluid to be drawn through the passage of the nanospray emitter for discharge from the outlet orifice.
Yet another aspect of the present invention relates to a method for making a durable nanospray emitter. This method includes the steps of providing a nanospray emitter body comprising a fluid inlet, an outlet orifice, and a passage communicating between the fluid inlet and outlet orifice; and casting a thin film of an electrically conductive polymer coating onto at least a portion of the nanospray emitter body.
A still further aspect of the present invention relates to a method of forming a nanospray of a solution which includes passing a solution including an analyte through a nanospray emitter of the present invention under conditions effective to electrically charge the solution in a manner to emit the solution from the emitter in the form of a nanospray.
Another aspect of the present invention relates to a method of analyzing a solution for an analyte in the solution. This method includes passing a solution including an analyte through a nanospray emitter of the present invention under conditions effective to electrically charge the solution in a manner to emit the solution from the emitter in the form of a nanospray, and then analyzing the nanospray emission in a manner suitable to detect an analyte present in the solution.
The conductive polymer coatings on emitters of the present invention are a significant departure from previously existing nanospray emitter coatings, all of which are based on metals. The conductive polymer-coated nanospray emitters of the present invention overcome the most problematic limitations of metallized emitters: they provide long-term durability while being simple to coat. With respect to a preferred embodiment, polyaniline (xe2x80x9cPANIxe2x80x9d) coated emitters are desirable because the PANI coating is resistant to corrosion (Wessling and Posdorfer, 1999) while maintaining high mechanical stability and antistatic properties (Triveldi and Dhawan, 1992). No PANI contamination is observed in the ESI spectra. Moreover, in its conductive form PANI is optically transparent (green), allowing for direct viewing of the ESI sample, and possesses outstanding adherence to glass properties (Manohar et al., 1991). PANI-coated nanospray emitters are relatively simple to produce and are highly resistant to electrical discharge. The sensitivity enhancement of PANI-coated nanospray emitters is similar to that of gold-coated emitters vs. normal ESI for tested analytes. Because of the significantly greater durability for the PANI coated emitters of the present invention, the emitters are available for use in analyzing, e.g., via CE-MS, highly complex biological matrices including, without limitation, drugs and their metabolites, neuropeptides, neurotransmitters, and biomolecules from complex biological fluids and tissues.